Apolipoprotein B (apo B) is the principal protein of the cholesterol-carrying low density lipoproteins (LDL) and is the determinant for cellular recognition and uptake of LDL by the high affinity LDL receptor. Binding of apo B to LDL receptors results in internalization and degradation of LDL, promoting the clearance of LDL from plasma and regulating intracellular cholesterol handling and biosynthesis. Post-secretory modification of LDL by oxidation, glycation or glycoxidation diminishes this high affinity LDL-receptor-mediated uptake and degradation (Klein et al, Diabetes 44:1093, 1995; Gugliucci et al, Scand J Lab Clin Invest 53:125, 1993). On the other hand, such modifications of apo B promote internalization by alternative receptors of monocyte-marcrophages that give rise to cholesterol-laden foam cells (Klein et al, Metabolism 38:1108, 1989; Klein et al, Diabetes 44:1093, 1995; Brown & Goldstein, Nature 343:508, 1990). Additionally, glycation alters the rate of clearance of LDL in vivo and interferes with intracellular handling of cholesterol and regulation of its synthesis, promoting increased serum cholesterol concentrations (Lopez-Virella et al, Diabetes 37:550, 1988; Steinbrecher & Witztum, Diabetes 33:130, 1984, Lyons et al, Diabetologia 30: 916, 1987; Klein et al, Diabetologia 33:299, 1990).
Glycated apo B is formed from the condensation reaction between glucose and reactive epsilon amino groups in the apolipoprotein, yielding an amino-deoxyfructose derivative in stable ketoamine linkage known as an Amadori product. It has been proposed that the Amadori product can give rise to a heterogeneous group of poorly defined advanced glycation end products (AGE) resulting from various rearrangement, dehydration and polymerization reactions. Prior art has suggested a pathophysiologic role of AGE-modified LDL in the pathogenesis of elevated LDL-cholesterol and vascular disease, and that inhibition of AGE-crosslink formation might be a beneficial treatment (Brownlee et al, Science 232:1629, 1986; New Engl J Med 318:1315, 1988; Vlassara, J Lab Clin Med 124:19, 1994; Bucala et al, Proc Natl Acad Sci 90:6434, 1993; Proc Natl Acad Sci 91:9441, 1994). However, recent experimental work as cited above indicates that apo B modified by Amadori products contributes to hypercholesterolemia and vascular dysfunction. Further, glycated LDL principally exists in vivo as the Amadori product and its concentration is elevated in people with increased risk for vascular disease (Cohen et al, Eur J Clin Chem Clin Biochem 31:707, 1993; Tames et al, Atherosclerosis 93:237, 1992).
The deleterious effects of Amadori-modified apo B make it desirable to have the means to prevent the attachment of glucose to apo B lysine-amino groups in vivo and thereby lower circulating concentrations of modified LDL. It would also be desirable to reduce plasma LDL-cholesterol levels by lowering concentrations of Amadori-modified LDL, thereby promoting LDL cholesterol clearance. Such means would reduce hypercholesterolemia and beneficially influence vascular dysfunction by mechanisms different from those disclosed in the prior art which relate to agents that lower cholesterol levels by inhibiting gastrointestinal absorption or cholesterol synthesis, or that prevent cross-linked AGE-modified LDL. Such means could be achieved with compounds that bind to apo B in a manner that effectively interferes with the modification of free lysine amino groups and thereby inhibit nonenzymatic glycation.
One manner in which post-secretory modification of apo B might be achieved is with compounds that prevent condensation of glucose with lysine amino groups. Acetylsalicylic acid (aspirin), by virtue of rapid acetylation of epsilon amino groups, can competitively inhibit this reaction (Rao and Cotlier, Biochem Biophys Res Comm 151:991, 1977; Rendell et al, J Lab Clin Med 107:286,1986). However, the impact of widespread protein acetylation is unknown. Moreover, the glycation-inhibiting activity of aspirin is relatively weak and potential therapeutic benefits that might be ascribed to this activity are limited by the rapid hydrolysis and short half-life of acetylsalicylic acid in the blood and by side effects anticipated at doses required to inhibit glycation in vivo (Costello and Green, Arth Rheum 25:550, 1982; Rowland and Riegelman, J Pharm Sci 57:1313, 1968). Other compounds which lack acetyl groups but bind to albumin in a manner that effectively interferes with the condensation of glucose with free lysine amino groups would be more desirable.
In an in vitro experiment van Boekel et al (Biochim Biophys Acta 1120:201, 1991) reported that 2,[2,6-dichlorophenyl-amino]benzene acetic acid (diclofenac) in concentrations of 1-5 mM could reduce the amount of carbohydrate-modified protein after incubation of commercially purchased powdered albumin with 5 mM glucose-6-phosphate (G-6-P). However, van Boekel et al did not examine whether this compound influences carbohydrate attachment to apo B. It is well know that albumin and apo B are proteins that have completely different primary, secondary and tertiary structures. It is also well known that the interaction with, or binding of small molecules to proteins is idiosyncratic and unpredictable. Thus, there is no expectation from the van Boekel reference that this compound could prevent modification of apo B either in vitro or in vivo simply because of impeded attachment of G-6-P to albumin in vitro.
Van Boekel et al conducted only in vitro experiments, only used G-6-P as the modifying substance, and only when the compound was present at 5 mM or more concentration. This concentration of the compound which was required to inhibit G-6-P attachment in vitro, and the composition and concentration of the substance (G-6-P) used to modify the protein, do not represent in vivo conditions. It is well known that although various reducing sugars such as G-6-P can condense with protein amino groups in vitro, the concentrations required vastly exceed those found of such sugars in vivo and that such reducing substances modify proteins in ways that are not representative in chemistry or nature of those which occur in vivo. It is therefore concluded that the compound would not be effective in inhibiting protein glycation in vivo since the primary sugar present in the circulation is glucose, and since the concentrations of the compound required to inhibit glycation in vitro would be toxic if given to living subjects. Diclofenac is usually administered in daily amounts of 100-200 mg. The peak plasma levels obtainable after such dosages are 1-2 .mu.g/ml, which is equivalent to 3-6 .mu.M. This is 1000-fold less than the concentration found by van Boekel et al to achieve any inhibition of albumin glycation in vitro, leading to the conclusion that dosages of 100-200 mg would have no effect on the glycation of any protein in vivo. Van Boekel et al additionally concluded that, because the compound binds to albumin, its concentration in tissues or for preventing modification of other proteins would be too low to be of import in disease states if administered in vivo. Van Boekel et al did not perform any in vivo experiments, does not afford any evidence that the compound could affect glycation in vivo, and contra-indicates the possibility that in vivo administration of therapeutically acceptable amounts of the compound could lower concentrations of modified apo B in living human subjects or could beneficially influence hypercholesterolemia or vascular dysfunction. Additionally, van Boekel et al emphasize that AGE, not Amadori products, are important in vascular disease, leading one to conclude that reducing glycated apo B per se would be without salutary effect in vivo on hypercholesterolemia or vascular disorders. Gamache et al (U.S. pat. No. 5,643,943) have reported the use of esters and amides of pharmaceutical compositions containing an anti-inflammatory and an anti-oxidant moiety for treating inflammatory vascular disorders. Gamache et al teach that a two-pronged approach is necessary in these formulations; namely, the combination of anti-inflammatory and anti-oxidant activity, and that these moieties be covalently linked by an amide or ester bond. The Gamache reference stipulates that compounds that fulfill these requirements are of the formula A--X--(CH.sub.2).sub.n --Y--(CH.sub.2).sub.m --Z wherein A is a nonsteroidal anti-inflammatory agent (NSAIA); A--X is an ester or amide linkage derived from the carboxylic acid moiety of the NSAIA, wherein X is O or NR; R is H, C.sub.1 -C.sub.6 alkyl or C.sub.5 -C.sub.6 cycloalkyl; Y is O, NR.sub.1 C(R).sub.2, CH(OH) or S(O).sub.n ; n is 2 to 4 and m is 1-4 when Y is O, NR, or S(O).sub.n ; n is 0 to 4 and m is 0 to 4 when Y is C(R).sub.2 or is not present; n is 1 to 4 and m is 0 to 4 when Y is CH(OH); n is 0 to 2; and Z is selected from the group of compounds consisting of the formula a, b, c, d or e taught by Gamache et al. Thus, Gamache et al conclude that diclofenac alone, without any amide or ester linked moiety, is useless in the treatment of hypercholesterolemia or vascular disease, and relates only to the synthesis of compounds combining an anti-inflammatory and an anti-oxidant moiety.
The present invention discloses the novel and unexpected discovery that therapeutically acceptable amounts of diclofenac inhibit the formation of glycated apo B in vivo, reduce hypercholesterolemia, and prevent the development of vascular disease which is manifested by increased urine protein excretion in human subjects.